Measurements of enzyme activity serve as important tools for the diagnosis of disease processes and for basic research. We are seeking to develop new substrates for enzymes that improve the ability to measure or the specificity of measuring enzyme activity. We have found that linking small chromogenic and fluorogenic substrates to polymer molecules serves as an approach to prepare substrates with large molecular size. Initial results are described in Clinical Chemistry 2001;47:215-222. The molecular size of the substrates is determined primarily by the size of the attached polymer so that it is adjustable. This permits substrate size to be analyzed as an independent variable, and it serves as a means of assessing the steric hindrance of the active sites of enzymes. The large synthetic substrates serve as better models of natural substrates that are of large molecular size such as the protein substrates of physiological proteinases and the large starch substrates of amylase. Use of large substrates appears to distinguish free protease molecules from those complexed with the inhibitor alpha-2-macroglobulin. Thus, these substrates appear to have utility for the more accurate measurement of the functional activity of plasma proteinases such as thrombin.